Animal feed composition

ABSTRACT

The present invention relates to an animal feed composition that comprises free indole acetic acid (free IAA) or a derivative thereof. The invention also relates to a method for enhancing animal growth by feeding the animal with a composition according to the invention. The invention also relates to the use of free IAA or a derivative thereof in a method of therapy of animals in need of a growth-promoting treatment, such as immunocompromised animals, animals with a growth deficit or slow growing animals. The invention also relates to the use of free IAA or a derivative thereof for the preparation of a therapeutical composition for increasing the growth rate and/or the feed conversion rate and/or the immunity of animals in need of such a treatment, in particular immunocompromised or slow growing animals. A composition according to the invention may preferably be in the form of a food or feed supplement.

FIELD OF THE INVENTION

The present invention relates to an animal feed composition thatcomprises free indole acetic acid (free IAA) or a derivative thereof.The invention also relates to a method for enhancing animal growth byfeeding the animal with a composition according to the invention. Theinvention also relates to the use of free IAA or a derivative thereof ina method of therapy of animals in need of a growth-promoting treatment,such as immunocompromised animals, animals with a growth deficit or slowgrowing animals. The invention also relates to the use of free IAA or aderivative thereof for the preparation of a therapeutical compositionfor increasing the growth rate and/or the feed conversion rate and/orthe immunity of animals in need of such a treatment, in particularimmunocompromised or slow growing animals. A composition according tothe invention may preferably be in the form of a food or feedsupplement.

BACKGROUND OF THE INVENTION

Limited supply of conventional food protein is a major problem facing arapid increase in world population. Of particular importance is theproduction of animals that contain proteins having essential amino acidsrequired by humans. Due to limited production facilities and lack ofimprovement in production technology, increase in animal proteinproduction does not seem to proportionate the increase in worldpopulation.

Therefore, it appears highly desirable to improve the productivity ofanimal protein production. One of the means to improve the productivityis to develop feed compositions that enhance animal growth.

It is known that the growth rate of animals can be accelerated byadministration of certain classes of substances such as antibiotics,surfactants and estrogens. The administration of each of these classesof substances had disadvantages, however, which have prevented theiruniversal acceptance. Thus, it is believed that antibiotics andsurfactants, while effective under certain conditions, principally actto suppress diseases and do not elicit a true growth response. It is forthat reason that such use of antibiotics will be limited in Europe,whereas it is under debate in the USA and Asia.

Also, the use of estrogens as growth-promoting agents presents certaininherent difficulties and hazards. Thus, estrogens frequently downgradethe animal to which they are administered. Another disadvantage is thatsome of the estrogenic material may remain in the edible portions of theanimal and could presumably produce an adverse effect on an individualwhen consumed. Further, the known substances have principally beenadministered by injection or implantation procedures, which are bothcostly and time-consuming and are ofyen disliked by consumers.

SUMMARY OF THE INVENTION

An object of the invention is to provide an animal feed composition.Another object of the invention is to provide a feed composition thatwould enhance animal growth. A further object of the invention is toprovide a method for enhancing animal growth by feeding the animal withthe feed composition. Another object of the invention is to provide amethod for the preparation of an animal feed composition. Other objects,features and advantages of the invention will become apparent as theinvention is more fully disclosed herein below.

It has now been found that the disadvantages of the prior art substancesand methods can be overcome, and that the growth of non-human animalscan be enhanced by orally administering free indole acetic acid (freeIAA) or a derivative thereof to animals in conjunction with their normalfeed or drinking water.

DETAILED DESCRIPTION OF THE INVENTION

Free IAA and its derivatives are known compounds. free IAA is anaturally-occurring plant growth phytohormone which has been extensivelystudied. In plants, most of the IAA occurs in a conjugated form (Slovinet al. 1999, Biochemistry and molecular biology of plant hormones,Elsevier, Amsterdam. P115-140), either conjugated to sugars via esterlinkages or to amino acids and peptides via amide linkages.

The term “free IAA” is used herein to indicate that the free IAA is inthe free or acid form, whereas the term “conjugated IAA” refers to IAAthat is conjugated via ester linkages or via amide linkages.

As long ago as 1956, the effects of free IAA on humans were studied, andit was shown that single doses of 0.1 g/kg were non-toxic (Mirsky A andDiengott D, Hypoglycemic action of indole-3-acetic acid by mouth inpatients with diabetes mellitus, Proc. Soc. Exp. Biol. Med. 93:109-110,1956). In 1964, it was found that photo-oxidation products offree IAA acted as growth inhibitors of micro-organisms (Still C,Fukuyama T and Moyed H, Inhibitory Oxidation Products of Indole-3-aceticacid, J. Biological Chemistry, 240,6,2612-2618, 1964).

Also, the medical use of free IAA and some of its derivatives haspreviously been described. EP 1,296,676 describes the use of free IAA asa pharmaceutical, in particular for treating neoplastic disease inhumans. WO 02/080906 describes the use of free IAA for treatingendometriosis in women. Nachson et al. (Food and Chemical Toxocology 41,745-752) reported the effect of some free IAA derivates(indole-3-carbinol and 3,3′-diindolylmethane) on the proliferation andinduction of apoptosis in human prostate cancer cell lines whereasRossiter et al. (Bioorganic & Medicinal Chemistry Letters, 12,2523-2526) as well as Folkes et al. (Biochemical Pharmacology 63,265-272) described the use of free IAA and some derivatives inenzyme-prodrug directed cancer therapies.

It has now been unexpectedly found that free IAA or a derivative thereofeffects a remarkable growth-promoting response in non-human animals,particularly farm animals such as fish, poultry, cattle and swine or petanimals such as horses, cats, dogs, rabbits and fish. The inventiontherefore provides a method of raising non-human animals byadministering free IAA to the animal, preferably via a feed.

The free IAA or a derivative thereof may be fed to the animal and ismost effective when administered within clearly defined concentrationsin the animal feed and/or water. Injection and/or implantation isunnecessary and the animals orally ingest the free IAA or a derivativethereof of their own free choice with their feed.

According to an embodiment of the invention, an animal feed compositionthat enhances animal growth is provided which comprises free IAA or aderivative thereof. Such an animal feed composition can be aconventional feed composition supplemented with free IAA or a derivativethereof in a concentration sufficient to allow a daily intake of between25 and 1000 microgram per kg life weight per day (ug/kg LW/day). Such afeed composition may then contain between 0.2 mg and 10 g free IAA or aderivative thereof per kg feed.

Particularly good results were obtained when directly feeding to ananimal a feed composition containing more than 0.24 mg free IAA or aderivative thereof per kg feed, such as a feed composition containingmore than 0,30 mg free IAA or a derivative thereof per kg feed, or afeed composition containing more than 0,40 mg free IAA or a derivativethereof per kg feed, such as more than 0,6, 0.8, 1, 5, 10, 20, 30, 50,100, 200 or 500 mg free IAA or a derivative thereof per kg feed.

The invention therefore relates to an animal feed composition comprisingmore than 240 microgram of free IAA or a derivative thereof perkilogram.

The upper limit of the concentration of free IAA or a derivative thereofis for a large part determined by the intended use. The feed compositionmay be in the form of a feed or food additive that has to mixed into theanimal feed at a convenient rate. In this case the concentration of freeIAA or a derivative thereof may be as high as 10%, i.e. 100 g/kg. Thefeed composition may also be in the form of a ready to use mixture. Inthat case the upper limit is determined by the desired dose for theanimal, once the daily intake of feed of the animal is known. In, dailypractice, feed compositions containing 1, 2, or 4% (10 to 40 g/kg) freeIAA or a derivative thereof proved to be most practical for storage inthe form of a stock feed additive. A preferred storage form of a feedcomposition to be mixed into a ready to use feed or into a feed additiveis therefore a mixture of about 1-4% free IAA or a derivative thereofwith other dry material suitable for ingestion.

The invention therefore relates to a feed composition comprising up to40 g of free IAA or a derivative thereof per kilogram.

Preparations containing between 100 mg and 1000 mg, such as 500 mg freeIAA or a derivative thereof/kg feed additive were most practical whenused to correctly dose the free IAA or a derivative thereof into theready to use feed composition.

The invention therefore also relates to a feed composition comprisingbetween 100 and 1000 mg of free IAA or a derivative thereof perkilogram.

A skilled person will appreciate that the amount of free IAA in theready to use feed has to be adjusted in order to supply the animal withan effective amount of free IAA. In order to adjust the free IAAconcentration in the feed so that a certain daily intake of free IAA isachieved, an estimate has to be made of the feed intake of an animal oranimal group. A skilled person is aware of the feed intake of a(particular kind or group of) animal(s), usually the feed intake per dayis between 0,5 and 10% of the body weight of the animal, with occasionalexceptions as high as 20% such as for instance in young chickens. Ananimal will normally ingest twice the unit volume of water as it willingest feed. Accordingly, an animal will ingest the same amount of freeIAA when fed with a feed composition containing 10 milligram free IAA/kgas it will by drinking water with a concentration of 5 mg/l or acombined concentration of 2.5 mg/l in water plus 5 mg/kg in feed.

An animal feed composition as used herein comprises a composition foranimal nutrition, in solid or in liquid form. Feed is the main source ofenergy and of nutrition for animals and is usually of animal or plantorigin. Thus feed may be defined as a substance with sufficientnutritional value to allow for growth and maintenance of adequate bodyconditions of an animal. In a typical embodiment, an animal feedcomposition consists of pellets, meal, grains, extruded or expandedgrains, tablets, powder or bolus forms. A particularly advantageous feedcomposition comprises a foodstuf selected from the group consisting ofdry forages and roughages, energy feeds, protein feeds, mineral feeds,vitamin feeds, yeast products, normal premix, cornmeal, cotton seedwheat gluten, maize silage rutabaga, sugar beet pulp, apple pulp,ryegrass, fescue grass, alfalfa feed concentrate and feed supplement.The free IAA or a derivative thereof may be mixed with any suitable basefeed material, such as rape seed, cotton seed, soybean, fish meal, wheatbran, wheat feed meal, minerals, vitamins and binders or prepared as apremix with, for example amino acids, salts, phosphorous or cornmeal. Inone particularly advantageous embodiment the feed or feed additive is ina form and/or composition approved by a governmental institution such asthe FDA, the US dept. of agriculture or the Canadian Food InspectionAgency. In Europe, the Task Force on animal feeding of the CodexAlimentarius Commission (CAC) as well as the Animal Welfare Act (AWA)provide definitions of animal feed. In one advantageous embodiment, theinvention concerns an animal feed within the definition of “animal feed”in section 201(x) of the FFDCA, supplemented with free IAA.

Free IAA or a derivative thereof is conveniently incorporated directlyin the animal feed and/or water. Any suitable method for dispersing thematerial in the feed can be used. The amount of free IAA or a derivativethereof added to the animal feed and/or water may be varied within thelimits stated in order to obtain the maximum benefits.

The invention therefore comprises a method for the preparation of animalfeed comprising the steps of providing an animal feed stuff and mixingsaid feed stuf with an effective dose of free IAA.

The composition according to the invention may be in the form of acapsule, but other dosage forms, preferably oral dosage forms, such astablets, suspensions, emulsions, fluids, powders, lozenges, pastilles,pills, etc., are also possible. The composition may for example take theform of a feed supplement, a feed additive or a pharmaceuticalcomposition.

In accordance with the present invention, there is also provided amethod for promoting the growth of non-human animals and/or improvingthe feed efficiency and/or the feed conversion rate and/or the immunityof a non-human animal that comprises administering to the animals ananimal feed composition according to the invention. Comprised herein isa method wherein a liquid such as water containing more than 120 ug freeIAA or a derivative thereof per litre fluid is administered to theanimals, such as 240, 500, or more than 1000 ug/l.

Feedstuffs and/or water supplemented with free IAA or a derivativethereof according to the invention are particularly suitable for thecommercial rearing of farm animals such as fish, cattle, poultry andswine. They may also be used for pet animals such as rabbits, horses,birds such as pigeons, fish such as koi fish, cats and dogs. Byutilising such supplemental diets, it is not only possible to greatlyaccelerate the growth of non-human animals but also the efficiency offeed conversion (i.e. the number of kg of feed needed to produce one kggain in animal weight) is greatly increased, resulting in greatereconomic benefits.

The animal feed composition and methods according to the invention mayalso be applied to animals that have a growth deficit. In animalexperiments it was shown that the weight of poorly growing animalsincreased to normal levels when fed with free IAA or a derivativethereof. These experiments are illustrative of the fact that thecompounds and methods of the invention lead to an increased appetiteand/or improved feed conversion.

The term growth deficit in this respect is to be regarded as a growththat substantially lacks behind the normal growth of the species. Suchanimals exhibit a life weight that is more than 10%, such as more than25, 40, 60 or 80% below the median of the normal weight distribution ofanimals with the same age within the species.

The animal feed composition and methods according to the invention mayalso be applied to immunocompromised animals. Immunocompromised animalsare herein defined as non-human animals with an impaired or weakenedimmune system. Such animals are usually characterised by a lowered levelof IGF-1 in their serum. Lowered IGF-1 serum levels are herein definedas levels of IGF-1 that are more than 10% below the normal median of thehealthy subjects in the species, such as more than 25%, 40%, 60% or morethan 80%. A weakened immune system is often the cause of an elevateddeath rate caused by disease or adverse living conditions.

The invention is also useful for the treatment of animals showingvarious other indications that are associated with a lowered serum levelof IGF-1. It is therefore a further object of the present invention toprovide a means for increasing the IGF-1 serum level in a non-humananimal suffering from a condition associated with a lowered IGF-1 serumlevel.

This is achieved according to the invention by administering free IAA ora derivative thereof to a non-animal in need of such a treatment.Accordingly, the invention thus relates to the use of free IAA and aderivative thereof for the preparation of a therapeutical compositionfor increasing the growth rate and/or immunity in immunocompromisedanimals and/or animals with a growth deficit. This may lead to anincreased body weight of the animal and/or a reduced death rate causedby diseases or adverse living conditions.

It was found that for the various indications as exemplified herein,there are optimal amounts of free IAA or a derivative thereof to beadministered. In general, in order to further increase the growth rateof healthy and normal growing animals, a daily intake of free IAA ofbetween 25 and 1000, more in particular between 50 and 500 ug/kg LW/dayis sufficient. Particularly good results may be obtained whenadministering 150-500 ug free IAA/kg LW/day. As exemplified hereinafter, African catfish showed a remarkably improved feed conversion rateof 7% when an average of 218 ug free IAA/kg LW/day was administered.Healthy poultry gained 18% more weight in 5 weeks when fed with a doseof 400 ug free IAA/kg LW/day. Comparable results were obtained in cattleand swine farms.

In order to increase the growth rate of animals that exhibit a growthdeficit (in particular farm or pet animals), a slightly higher dose isoften required. Particularly good results were obtained with a dose of50-1000 ug free IAA/kg LW/day, in particular 250-1000 ug free IAA/kgLW/day, more in particular 400-1000 ug free IAA/kg LW/day such as 500 or750-1000 ug free IAA/kg LW/day during a short period of time such asless than 6 weeks, 4 weeks but more in particular less than 3 weeks,such as 2 weeks.

Optimal dosage of free IAA or a derivative thereof may be determinedempirically, and may, within the limits defined herein, depend to someextend on the particular type of feed, species and farming conditions. Aperson skilled in the art knows how to set up a schedule for such dosingexperiments, the experimental design of example 7 may be helpfultherein.

The key compound of the invention is thus free indole acetic acid.However, equivalent result may be achieved by using derivatives of freeIAA. Derivatives of free IAA are herein defined as compounds that leadto an increased level of free IAA in the body of the non-human animal incomparison to the level of free IAA in the same animal body prior toadministration of the compound. These derivatives can be divided intovarious categories.

One category of derivatives of free IAA is conjugated IAA. ConjugatedIAA may appear in the form of IAA conjugated via ester linkages, forexample to sugars such as for example IAA-glucose, IAA-alfa-asparticacid 1N-glucoside, IAA-inositol, IAA-myoinositols or IAA linked tovarious other carbohydrates. IAA may also be conjugated via amidelinkages for instance to amino acids and peptides. Examples thereof areacetamide, alfa-leucine, alfa-alanine, alfa-aspartate (most importantconjugate of IAA in plants), alfa-glutamate, alfa-lysine, alfa-glycine,alfa-valine, alfa-phenylalanine or slow release amide conjugates withlysine or tryptophan. Conjugation with peptides is common, whereas alsoconjugates with other amino acids occur in different plants. Inaddition, this group comprises 3-acetonitrile derivatives, which easilyare converted into the corresponding acid, like indole-3-acetonitrilethat decomposes in free IAA both chemically (under basic conditions) andenzymatically (by nitrilase activity

In order to be useful in the present invention this conjugated IAA mustbe converted to free IAA. This may be achieved by simultaneous admissionof enzymes such as esterases, amidases or nitrilases to the animal. Thismay for instance be achieved by admixing enzymes together with theconjugated IAA into the animal feed composition.

The invention thus relates to an animal feed composition as describedabove, additionally comprising an enzyme capable of converting aderivative of free IAA into free IAA.

However, the conjugated IAA may also be added to the feed and/oradministered to the animal as such, thereby by relying on the enzymeactivities naturally present in the circulation or gastro-intestinaltract of the animal for the conversion of conjugated IAA into free IAA.

Free IAA is liberated from the amides by amidases (amidohydrolysases).Free IAA may be released from the glucosides by glucosidases. In plants,considerable amounts of conjugated IAA can be present, which can beliberated either by enzymatic hydrolysis (such as glucosidases oramidases) or chemical hydrolysis. Of the total IAA pool in plants, amidelinked IAA in general constitutes 90%, whereas approximately 10% isester linked and less than 1% is free IAA. In plants, levels offree+bound free IAA as high as about 1.2 ug/g Dry Weight may be foundsuch as in 9 days old Arabidopsis. Later in the life cycle of the plantthese levels drop. A maximum of about 1% of this is free IAA.

In plants also a variety of hydroxylated, phosphorylated, methoxylated,N-oxides and N-methylated indole derivatives can be found. Thesecompounds can also be converted into free IAA, either in the stomach,gut, liver or elsewhere in the body. This conversion can be enzymatic orchemical. Thus the invention also relates to these compounds that can beconverted to free IAA, either directly or indirectly through metabolicconversion. These so-called precursors are for example 4-hydroxy-IAA,4-methoxy-IAA, 5-hydroxy-IAA, 5-methoxy-IAA, 6-hydroxy-IAA,6-methoxy-IAA, 7-hydroxy-IAA, 7-methoxy-IAA.

Furthermore, the term derivative may also comprise IAA with othersubstituents, compounds that may be either naturally occurring orsynthetic. The term “naturally occurring” also encompasses the result ofmetabolism by living cells such as plants, micro-organisms, mammaliancells and the human or animal body. In nature halogenated indolealkaloids can be found, particularly in marine organisms (i.e.6-bromoindigotin). All types of substituents can synthetically beintroduced on the aromatic ring, e.g. methyl, amino, nitro, fluoride,chloride, bromide, and iodide on the positions 4, 5, 6 and 7. Thesecompounds may all be used to increase the level of free IAA in ananimal.

The term derivatives may also encompass precursors from which free IAAand analogues as listed above could be formed, such as tryptophan,4-hydroxytryptophan, 4-methoxytryptophan, 5-hydroxytryptophan,5-methoxytryptophan, 6-hydroxytryptophan, 6-methoxytryptophan,7-hydroxytryptophan7-methoxytryptophan, hypaphorine, tryptamine,4-hydroxytryptamine, 4-methoxytryptamine, psilocin (4-hydroxy, dimethyltryptamine), psilocybin (4-phosphate, dimethyl tryptamine), baeocystin,serotonin (5 hydroxytryptamine), 5-methoxytryptamine, bufotenine(dimethylserotonine), O-methylbufotenine, melatonin (5-methoxy,acetamide function on tryptamine NH₂), 6-hydroxytryptamine,6-methoxytryptamine, 7-hydroxytryptamine, 7-methoxytryptamine. Othernaturally occurring precursors for free IAA formation are indole butyricacid and indole-3-pyruvate.

The term derivatives may also encompass compounds that are analogues ormetabolites of free IAA that may be converted back into free IAA. Thesecompounds also encompass the above mentioned 4-, 5-, 6- and 7-hydroxy-and methoxy-derivatives. These compounds are for example indole,indole-3-acetaldehyde, indole-3-ethanol, indole-3-aldehyde,indole-3-methanol, indole-3-carboxylic acid, 3-methylindole (skatole),indole-3-acetaldoxime, 3-aminomethylindole, N-methylaminomethylindole,Gramine (N-dimethylaminomethylindole).

The term derivatives may also encompass compounds with changed indolechromophore, such as indoxyls (indicans), indoleninones,3-methylene-2-oxindole, abrine, isotan B, isatin, indican, indigo,indurubin, indigotins,3-indolylmethyl (skatolyl), niacin and2-oxindole-3-acetic acid.

The term derivatives may also encompass compounds that are commonlyfound in plants or vegetable tissue, such as 3-methylene-2-oxindole,oxindole-3-methanol, oxindole-3-aldehyde, oxindole-3-carboxylic acid and3-methyloxindole.

Furthermore, the invention also relates to the use of conjugates (estersand amides) of other natural IAA derivatives such as 2-oxindolederivatives and 4-, 5-, 6- or 7-hydroxyderivatives: dioxindole-3-aceticacid, 3-O-beta-glucosyl-dioxindole-3-acetic acid7-hydroxy-2-oxindole-3-acetic acid-7′-O-beta-d-glucopyranoside,glucopyrasonyl-beta-1,4-glucopyranosyl-beta-1-N-oxindole-3-acetyl-N-asparticacid, glucopyranosyl-beta-1-N-oxindole-3-acetyl-N-aspartic acid,2-indolone-3-acetyl aspartic acid,3-(O-beta-glucosyl)-2-indolone-3-acetyl aspartic acid,3-hydroxy-2-indolone-3-acetyl aspartic acid, indole-3-glycerophosphate(decomposes in free IAA under basic conditions), indole-3-glycerol(decomposes into free IAA under basic conditions), glucosinolates, suchas indole-3-ylmethyl glucosinolate (glucobrassicin),4-hydroxyindol-3-ylmethyl glucosinolate (4-hydroxyglucobrassicin),1-acetyl-indol-3-ylmethyl glucosinolate (1-acetyl-glucobrassicin),1-methoxyindol-3-ylmethyl glucosinolate (neoglucobrassicin),4-methoxyindol-3-ylmethyl glucosinolate, (4-methoxyglucobrassicin), 1-sulfo-indol-3-ylmethyl (glucobrassicin-1 -sulfate) which are convertedinto indole derivatives by myrosinases (thioglucosidases).

In general, derivatives of free IAA are preferably molecules that can besynthesised into free IAA in one step, either by chemical synthesis orby enzymatic conversion. Examples of such derivatives areindole-3-acetaldehyde (IAAId) that can be converted into free IAA byaction of IAAId oxidase (AAO1) or indole-3-acetonitrile (IAN) that canbe converted into free IAA by nitrilases NIT1, NIT2, or NIT3 (Bartel etal., J. Plant growth Regul (2001) 20; 198-216). Alternatively,derivatives of free IAA may be molecules that can be synthesised intofree IAA in two steps. Examples of such molecules are indoleglucosinolate that involves the action of an enzyme known as myrosidase,indole-3-acetaldoxime (IAOx) and many other precursors that are nowapparent for a person skilled in the art. Alternatively, derivatives offree IAA may be molecules that can be synthesised into free IAA in threeor more steps.

The invention therefore relates to an animal feed composition comprisinga derivative of free IAA wherein the derivative can be converted intofree IAA in more than 3 steps, preferably in 3, more preferably in 2 andmost preferably in 1 step.

It may be apparent now for the skilled person that the dose of aderivative of free IAA in the animal feed is to be adjusted in order toyield free IAA concentrations in the animal body that correspond to theranges given herein for free IAA. This has to account for conversionsthat are mostly not entirely complete and losses during production whenprepared chemically. Concentrations of derivatives may therefore best beestablished empirically, the experimental set-up as outlined in example7 may be helpful in this respect. Consequently, when it is referredherein to a certain concentration of free IAA or a derivative thereof,it is meant that this is the concentration of free IAA or theconcentration of the derivative that yields this particularconcentration of free IAA in the animal. The skilled person will beaware of this and knows how to determine the proper concentrations ofderivatives with the help of the teachings as provided herein.

Free IAA is readily available as a commercial product. It may besynthesised chemically or prepared in a biological way. IAA producingmicro-organisms are widespread in nature. Yeast, fungi and many bacteriaas well as plants are known to convert precursors of IAA into free IAA.In addition to the L-tryptophan conversion by bacteria, alsoL-tryptophan independent biochemical routes towards free IAA aredescribed extensively (J. Plant Growth Regul (2001) 20: 198-216).

A well known bacterium, capable of producing free IAA is AzospirillumBrasilense (AB). At the end of the growth phase in a regularfermentation process, AB is able to convert L-tryptophan into free IAA.To increase the efficiency of this conversion, a small amount ofsynthetic free IAA may be added to the media. Via a feedback mechanism,AB increases the conversion of L-tryptophan into free IAA.

Final concentrations of 1 gram free IAA/liter culture broth are easy tomake, but even much higher concentrations are possible, depending on themicro-organism used.

After ending the fermentation the micro-organism may be lysed and apowder enriched in free IAA may be obtained by spray drying or any otherconvenient way of drying the culture broth. Other techniques may be usedto remove liquids partly or completely.

EXAMPLES Example 1 Source of free IAA

Free IAA or a derivative thereof may be obtained from any commercialsource. Alternatively, free IAA may be produced in a microbiologicalway.

To this end, Azospirillum brasilence Sp7 (ATCC) was obtained as an agarculture in a culture tube. LB medium was used to grow the strainovernight at 28° C. at 175 rpm. Glycerol was added to the culture up to10%, mixed and divided over Nalgene creovials and frozen at −80° C.Stocks were stored at −80 ° C. in creovials.

To prepare a seed culture of A. brasilence, one stock (1.2 to 1.8 ml)was thawed and added to 1 liter of LB medium and grown for about 20 h at28° C. and 175 rpm to an Optical Density (OD620 nm) of about 2.5.

A 10 litre fermentor was rinsed with water and the pH electrode wascalibrated. Nine litre of LB medium was prepared and 1 g/l L-Tryptophanand 0.1 g/l free IAA was added. The medium was entered into thefermentor together with 2 ml of anti foam. The fermentor was sterilisedfor 30 min at 121° C. After cooling down to 28° C., the O2 probe iscalibrated with N2 and O2, 0 and 100% air saturation respectively.

The seed culture is transferred to the fermentor via a flask and tubingwhich are separately sterilised in an autoclave. When the addition iscompleted the tubing and flask are removed and the fermentation isstarted with the following parameters: Stirrer speed 400 rpm Temperature28° C. Aeration 0.75 Nl/min PH 7

After 15 min a sample is taken to measure the OD620 nm and check the pH.Samples are taken at certain intervals to quantify the growth of A.brasilence. When the growth rate declined extra medium was added toensure that enough biomass was formed for the production of free IAA. Itwas found that the production of free IAA started when the active growthphase ended and continued for a prolonged period. The course of the freeIAA concentration was followed by LC-MS. When the concentration of freeIAA was at a level of about 1 g/l, the fermentation was terminated andthe cells were harvested and lysed by means of a nonojet homogeniser atabout 1400 bar. The remaining supernatant and the lysed cells weresterilised and spray dried to yield the desired product formulation.

Example 2 Growth Rate of Poor Growing Piglets can be Improved with FreeIAA

The trial was done in a well managed farm with 1000 sows of the DutchLand race. Although the farm is well run, the technical performance wasnot optimal. There were latent problems with mortality and with growthrates of the piglets. There were too many poor growing piglets, withouta clear underlying technical or veterinary reason. There was no clearpathology to be seen on the farm.

Three groups of piglets were randomly selected at day 1 of the trial.The first (control) group consisted of normal well growing piglets. TheB group consisted of 78 poor growers, these were treated with free IAA.The X group consisted of 52 poor growers that were not treated.

The B group received free IAA in the feed, starting on day 5 of thetrial. The feed for the B group was prepared by first mixing free IAAwith dextrose which was then mixed with the feed. For that purpose, 4gram of pure free IAA (Aldrich) was mixed with 96 gram dextrose and themixture was then dispersed in the feed. The pigs from the B groupreceived a dosage of 500 ug free IAA/kg LW/DAY (LW=life weight) whichcorresponds to 12.5 mg/kg LW/DAY of the 4% free IAA/dextrose mixture.

The piglets were weaned for two days at the start of the trial. Bloodsamples for IGF-1 measuring were taken from each group at day 5 of thetrial and at the end of the trial. Two pens (13 piglets) of the B and Xgroup, were weighed at day 5 and at the end of the trial. Quantificationof IGF-1 was performed using an immunoradiometric assay (IRMA)(DSL-5600ACTIVE™, DSL, Germany GmbH, Germany). Intra- and inter-assay variancewas: 4.0% and 9.2% for GH; 3.0% and 1.5% for IGF-1.

Already after one week of treatment, the farmer noticed a cleardifference between the group B and group X. The piglets in group B werelooking better, the bellies were better filled and the generalappearance of the piglets started to look better than in group X. Thisphenomenon became more pronounced when the treatment continued. Therewere less poor growers in the B group, skin and hair were looking muchbetter.

At day 1 of the trial there was no difference in IGF-1 levels betweenthe 3 groups (Table 1). IGF-1 levels were low, between 0.8 and 13.1 withan average of 4.6. There was no observable difference between the (wellgrow) control group and the (poor grown) groups X and B, probably due tothe stress of weaning.

At day 26 of the trial, IGF-1 levels were measured again. The B groupwas on the level of the healthy control group (25.3 vs. 23.6 nmol/l)which was clearly higher than the non-treated group X (17.2 nmol/l).TABLE 1 IGF-1 measurement [nmol/l] Day 1 Day 26 Control group 1 0.8 14 24.3 16.6 3 7.9 25.4 4 6.1 30.3 5 3.9 31.7 total 23 118 average 4.6 23.6Treated group B 1 3 25.2 2 2.7 28.9 3 6.1 25.5 4 5.3 29.9 5 5.7 16.9total 22.8 126.4 average 4.56 25.28 Non-treated group X 1 2.9 11.3 2 4.321.6 3 1.6 died 4 13.1 20.8 5 1.3 15.1 total 23.2 68.8 average 4.64 17.2

Pen B 3L(eft) grew on average 850 grams more then their neighbours frompen X 4 L(eft). Pen B 3 R(ight) gained on average as much weight astheir neighbours from pen X 4R(ight), but weighed 310 grams less at thestart of the trial. On average, the treated group gained (for the pensthat were weighed) almost 0.5 kg more over a period of 21 days (table2). TABLE 2 Weight (kg) Weight (kg) Weight gain at day 5 at day 26 (kg)Pen nr Total (kg) Average Total (kg) Average Average B 3L 86 (n = 13)6.62 150 (n = 11) 13.64 7.02 B 3R 88 (n = 13) 6.77 167 (n = 13) 12.856.08 X 4L 80 (n = 12) 6.67 141 (n = 11) 12.82 6.15 X 4R 92 (n = 13) 7.08171 (n = 13) 13.15 6.08

After the treatment was stopped, the piglets from group B continued toperform better than group X. Piglets from group B started to look betterthan the non-treated group and grew better than the untreated group.These characteristics are clearly correlated with a higher IGF-1 levelin the treated groups.

The results of this trial confirm that a single treatment for 14-21 dayswith 500 ug free IAA/kg LW/DAY effectively restores IGF-1 levels in poorgrowing piglets and promotes growth up to a level of normal welldeveloped piglets. As a result of this treatment pigs have caught up thelost growth and have done well during the fattening period, without theneed of continuing the treatment.

Example 3 Growth of Healthy Laying Hens can be Improved with Free IAA

In this example normally growing animals were treated with free IAA.Growing laying hens of 10 weeks old were selected in the flock to createtwo groups of ten hens:

-   Group GB: 10 normally growing hens, not treated-   Group GNA: 10 normally growing hens, treated with free IAA

The treated birds were force fed daily with a capsule with 400 ug freeIAA/kg LW/DAY which corresponds to 10 mg/kg LW/DAY of a 4% mixture offree IAA in dextrose. Treatment was continued until the hens startedlaying. The two groups were weighed every week.

From the first week on, an improved weight gain was seen in the treatedgroup when compared with the non-treated group. The improvement inweight gain was consistent over the first four weeks of the trial. Thegroup GNA was about 2 weeks in advance of the normal rearing schedule.

The last couple of weeks of the experiment were very stressful for thebirds, since it was very hot, and the birds were vaccinated against ILT.TABLE 3 Group GB Group GNA Average Average Average Average weight weightweight weight Week (gr) gain (gr) (gr) gain (gr) 1 690.5 740 2 802 111.5883 143 3 878 76 996 113 4 995 117 1095 99 5 1098 103 1222 127 totalweight 407.5 482 gain (gr)

The weight gain already after one week gave a clear indication that freeIAA has a beneficial effect on the growth rate of normal hens. It can beconcluded that free IAA has a beneficial effect on the growth of normalbirds, and that free IAA can speed up the normal rearing process anddeliver much stronger birds at the end of the rearing period. After 5weeks of trial, the birds who received free IAA in their feed were 18%heavier on average than the control group that did not receive free IAA.

Example 4 Performance of Poor Growing Laying Hens can be Improved withFree IAA

Poor growth of laying hens constitutes a big problem in rearing hens. Inthis example, poor growing birds were treated with free IAA. The flocksin the test farm did not grow uniformly, and about 10-15% of the birdshad a too poor growing performance.

Growing laying hens of 10 weeks old were selected in the flock to createthree groups of ten hens:

-   Group GB: 10 normally growing hens, not treated-   Group SB: 10 poorly growing hens, not treated-   Group SNA: 10 poorly growing hens, treated with free IAA

The treated birds were force fed daily with a capsule with 400 ug freeIAA/kg LW/DAY which corresponds to 10 mg/kg LW/DAY of a 4% mixture offree IAA in dextrose. Treatment was continued until the hens startedlaying. The different group were weighed every week.

From the first week on, an improved weight gain was seen in the treatedSNA group when compared to the non-treated SB group. The improvement inweight gain was consistent over the first four weeks of the trial. TheSNA group had caught back up to the schedule of normal growth and insome weeks outperformed the normal growing hens that were not treated ingroup GB.

The last couple of weeks of the experiment were very stressful for thebirds, since it was very hot, and the birds were vaccinated against ILT.TABLE 4 Group GB Group SB Group SNA Average Average Average AverageAverage Average weight weight weight weight weight weight WEEK (gr) gain(gr) (gr) gain (gr) (gr) gain (gr) 1 690.5 498.5 489.5 2 802 111.5 58586.5 598 108.5 3 878 76 685 100 718 120 4 995 117 790 105 885 167 5 1098103 907 117 995 110 total 407.5 408.5 505.5 weight gain (gr)

The weight gain already after one week gave a clear indication that freeIAA has a beneficial effect on the growth rate of hens with a growthdeficit. The group SNA shows the highest weight gain in this testperiod. It is concluded that free IAA may suitably be used in thetreatment of hens that have a growth deficit in order to have them catchup their “lost” growth.

It can also be concluded that free IAA does not seem to induce aresistance in hens to it's mode of action at the dosage of 400 ug freeIAA/kg LW/DAY.

So the results of this example indicate that the use of free IAA canbring poor growing hens back to the normal rearing schedule and preventloss of animals for normal production, and that free IAA can delivermuch stronger birds at the end of the rearing period.

Example 5 Survivors of Porcine Reproductive and Respiratory SyndromeVirus Fed with Free IAA

This experiment was performed with Belgian Land Race piglets that had ahistory of PRRSV. The piglets were weaned at four weeks and relocated attwelve piglets per pen. IGF-1 levels from three different groups weretested at the age of five weeks.

Group P

These were the pigs with the big problems. They were looking poor, had alow weight, bad colour and some of them had Staphylococcus infections.It was the firm belief of the farmer and the veterinarian that thesepiglets would not survive until the end of the fattening cycle. Bloodsamples were taken at random from five of the twelve piglets. TABLE 5Animal IGF-1 [nmol/l] at day 1 Piglet 1 2.3 Piglet 2 <0.5 Piglet 3 <0.5Piglet 4 2.0 Piglet 5 1.8

The results of table 5 indicate a severe impairment of the immune systemas indicated by low IGF-1 levels. A clear correlation exists betweenpoor growth and health and IGF-1 concentration. Each animal ofapproximately seven kilos was treated with 125 mg of a 4% free IAAcomposition comprising 5 mg free IAA and 120 mg NaCl and WPC 70 (WheyProtein Concentrate) for a period of ten days. The product was fed in atrough, mixed in liquid feed. All the piglets ate from the same trough.This dosage corresponds to a treatment with 715 ug free IAA/kg LW/day.

After ten days of treatment all twelve pigs were still alive and theircondition had dramatically improved. They all had a nice pink colour, nomore stiff hair, the ears were in normal position, no piglets sufferedfrom Staphylococcus infection anymore. They had gained very much inweight and in muscle growth and had caught up very much to the averageof the rest of the “normal” litter mates. All external signals for goodhealth were now present.

After ten days, blood samples were taken from five randomly chosenpiglets and their IGF-1 concentrations were determined. A code was addedfor the size of the animals (K=small; N=normal; Z=heavy). TABLE 6 AnimalIGF-1 [nmol/l] at day 10 Piglet PN 0.8 Piglet PK <0.5 Piglet PZ 4.2Piglet PK <0.5 Piglet PN <0.5

The dramatic improvement in health, weight and condition of the pigletswas not reflected in their IGF-1 levels. The average IGF-1 levels didnot rise during the first 10 days of the trial. We assume that at thattime all the extra IGF-1 production is still used in the process of theextra-growth. Yet, there is a clear link between individual weight andIGF-1 levels.

After 10 days the free IAA treatment was stopped and the animals wereheld on a normal diet. Another three weeks later blood samples weretaken again from 7 randomly chosen pigs from the group. TABLE 7 AnimalIGF-1 [nmol/l] at day 31 Piglet 1 30.0 Piglet 2 23.4 Piglet 3 65.1Piglet 4 13.7 Piglet 5 60.1 Piglet 6 36.9 Piglet 7 55.6

These results clearly show that the piglet's IGF-1 production was nowconsiderably increased, even though the treatment was stopped.

It may be concluded that the treatment with free IAA dramaticallyimproved the condition of the problem piglets. Their immune systemeliminated the Staphylococcus infections. This improvement was notimmediately (after 10 days) reflected in the IGF-1 serum concentration,but three weeks after the treatment was stopped, the IGF-1 level hadrisen to (almost) normal levels.

Group R

These piglets were looking the best. They received a special pre-starterstarter feed before weaning. Five piglets was chosen at random and theirIGF-1 levels were determined (table 8). TABLE 8 Animal IGF-1 [nmol/l] atday 1 Piglet 1 4.3 Piglet 2 1.0 Piglet 3 7.3 Piglet 4 22.2 Piglet 5 14.7

The special pre-starter seemed to boost the IGF-1 levels in this group.As from day 1, these piglets were fed a normal commercial piglet starterdiet, supplemented with the same dosage free IAA as group P. Group R wasgrowing normally. No particular signs were present. At day 10, bloodsamples were taken from six piglets selected at random. TABLE 9 AnimalIGF-1 [nmol/l] at day 10 Piglet RK 11.4 Piglet RN 11.4 Piglet RN 32.6Piglet RZ 18.7 Piglet RK 9.8 Piglet RZ 10.4

The average IGF-1 concentration in group R rose in two weeks from 9.9nmol/l to 15.72.

Group T

These were normal looking piglets. They received a regular pigletstarter feed before weaning TABLE 10 Animal IGF-1 [nmol/l] at day 1Piglet 1 2.7 Piglet 2 <0.5 Piglet 3 0.8 Piglet 4 0.5 Piglet 5 3.5

These piglets have normal growth, but less good then the R group. Thisis also reflected in the lower IGF-1 levels.

These piglets were kept on their original piglet starter feedsupplemented with the same dosage free IAA as groups P and R. Group Twas also growing normally without any particular symptoms. Blood sampleswere taken from 5 pigs selected at random and their IGF-1 concentrationwas determined. TABLE 11 Animal IGF-1 [nmol/l] at day 10 Piglet TZ 10.5Piglet TN 6.0 Piglet TN 3.1 Piglet TN 10.9 Piglet TZ 16.3

The average IGF-1 concentration in this group rose in 10 days from 1.6to 9.36 nmol.

Example 6 Piglets with Growth Deficit using Free IAA in the PrestarterFeed

It is generally accepted that animals that have lower weights in earlylife have a lower performance all over the production period. Thedifference in early growth can for a great deal be explained by adifference in individual sensitivity to stress and/or infectionpressure. This experiment was designed to study the effects of free IAAon the growth rate of healthy but poor growing pigs at an early stage oftheir life.

The test was carried out on a large commercial farm with 1400 sows inwhich poor growing piglets were selected. The selected piglets were notsuffering form any specific disease and piglets on the farm were testednegative for antibodies against PRRS (sows were vaccinated againstPRRS). 39 Piglets were selected from a group of 600 at day 21 of age, 7days before weaning. These piglets were at random divided in 3 groupsover 3 fostering sows until weaning. Two groups of piglets received freeIAA in the prestarter feed in the farrowing period at a dose of 500 ugfree IAA/kg LW/day. After weaning, free IAA was supplemented to these 2groups in the starter feed at the same dose until 14 days after weaning.The amount of free IAA added to the feed was based on the estimatedweights and feed intake. The third group received the same feed, butwithout free IAA.

The animals were weighed at weaning and 14 days later and mortality wasmonitored. The weight developments of the three groups are given intable 12. TABLE 12 Weight and growth of the piglets Number of animals ineach Average weight (kg) Average weight Day group Day 28 (weaning) Day42 gain (g/day) Group 1, 16 4.31 5.7 99 Control Group 2, 12 3.35 5.0 117free IAA Group 3, 11 4.22 6.2 141 free IAA

The groups treated with free IAA performed better than the controlgroup; both group 2 and 3 gained more weight than the control group.Free IAA clearly improved the performance of poor growing piglets. Inthe control group, 2 animals died versus none in the free IAA treatedgroups.

Poorly growing piglets (runts) are often taken out of production becauseof poor performance and anticipated high medication costs. Treatmentwith free IAA may contribute to save these animals, to lower theirmedication costs and to improve performance and economics of a farm.

Example 7 Effect of Different Concentrations of Free IAA on AfricanCatfish.

A number of 360 African catfish (Clarias gariepinus) of approximately 30grams each were used in this trial. One week after arrival, the fishwere spread randomly over 12 identical aquaria, 30 fish in eachaquarium. The temperature of the water was set at 25° C. andillumination schedule was 12 hour light followed by 12 hour darkness.The water circulation was 2 liter per aquarium per hour; the volume ofwater in each aquarium was adapted to the biomass. To get used to thefeed, the fish were fed the basic feed during two weeks prior to thestart of the trial.

Four groups were formed, each consisting of 90 fish divided over 3aquaria. One group received no treatment and served as a control. Theother three groups received free IAA treatment in three different dosesas indicated in table 13. Feed was prepared by mixing free IAA withbasic feed in the concentrations indicated in Table 13. There was afixed amount of feed given to the fish each day corresponding to 2.5% ofthe biomass. TABLE 13 Feed given (mg free Dose based on actual feedIAA/kg intake of 2.5% of live weight Group feed) (ug free IAA/kg LW/day)1 0 0 2 40 1360 3 16 544 4 6.4 218

The trial period lasted for five weeks and the fish were weighed everyweek. The average growth over the whole trial period is given in table14 below TABLE 14 Dose Group (ug free IAA/kg LW/day) Average growth(g/d) 1 0 4.96 2 1360 5.02 3 544 4.97 4 218 5.31

The results show that Group 4 (the dose of 218 ug free IAA/kg LW/day)gave a clear and significant improvement of 7.1% over the control at thesame feed intake. Because all fish received exactly the same amount offeed, this means that also the feed conversion rate was improved withthe same level. This is a significant increase for the fish industry andreflects the potential of free IAA in the stimulation of growth ofhealthy fish. The higher dosages did not show any clear positive ornegative response in comparison to the control. It may therefore beconcluded that an optimal dose for this kind of treatment is to bedetermined empirically.

Example 8 Preferred Stock Mixtures of IAA

A stock of a 4% free IAA feed stock additive was prepared. For thatpurpose free IAA (Aldrich) was mixed with 94% protamyl and 2% yeastextract. From that stock, 1,25% was added to a feed additive as detailedbelow in table 15. TABLE 15 Compound % kg 4% IAA stock 1.25 0.625Fe-sulphate 0.5 0.25 Protamyl 5 2.5 WPC 35 45 22.5 Vitamin C 2.5 1.25Maltodextrose 45.55 22.775 Vitamin E 0.2 0.1 SUM 100 50

The feed additive according to table 15 may be added to animal feed inappropriate amounts to ensure the desired dose to be administered to ananimal. It may be apparent that the concentration in the feed may varydepending on the daily intake of feed of the animal as well as on theanimal's weight. In order to supply an animal of 10 kg with a dailyintake of 500 ug free IAA per kg LW, an amount of 10 grams of themixture of table 15 may be mixed with the amount of feed that the animalingests per day.

1. An animal feed composition comprising more than 240 microgram of freeIAA or a derivative thereof per kilogram, wherein said derivative isselected from the group consisting of 4-hydroxy-IAA, 4-methoxy-IAA,5-hydroxy-IAA, 5-methoxy-IAA, 6-hydroxy-IAA, 6-methoxy-IAA,7-hydroxy-IAA, 7-methoxy-IAA and a compound that can be converted intofree IAA in one or more steps.
 2. A feed composition according to claim1 comprising up to 40 g of free IAA or a derivative thereof perkilogram.
 3. A feed composition according to claim 1 comprising between100 and 1000 mg of free IAA or a derivative thereof per kilogram.
 4. Afeed composition according to claim 1 additionally comprising an enzymecapable of converting the derivative into free IAA.
 5. A feedcomposition according to claim 4 comprising an aromatic ring wherein thearomatic ring is substituted on one or more of the 4, 5, 6 and 7position with methyl, amino, nitro, fluoride, chloride, bromide oriodide.
 6. A feed composition according to claim 1, wherein the feedcomposition is in the form of at least one of pellets, meal, grains,extruded or expanded grains, tablets, powder and bolus forms.
 7. Amethod for at least one of increasing the growth rate and improving atleast one of the feed efficiency, the feed conversion rate and theimmunity of a non-human animal, the method comprising administering tosaid animal an effective amount of a composition according to claims 1.8. A method, comprising: using free IAA or a derivative thereof, whereinsaid derivative is selected from the group consisting of 4-hydroxy-IAA,4-methoxy-IAA, 5-hydroxy-IAA, 5-methoxy-IAA, 6-hydroxy-IAA,6-methoxy-IAA, 7-hydroxy-IAA, 7-methoxy-IAA and a compound that can beconverted into free IAA for the preparation of a therapeuticalcomposition for stimulating the immune system in non-human animals inneed of such a treatment.
 9. A method, comprising: using of acomposition according to claim 1 for the preparation of a therapeuticalcomposition for at least one of stimulating growth and stimulating theimmune system in animals in need of such a treatment.
 10. A methodaccording to claim 8, wherein the free IAA or a derivative thereof iscapable of increasing the serum level of insulin-like growth factor 1(IGF-1).
 11. A method according to claims 10, wherein the animal has alowered level of IGF-1.
 12. A method according to claims 8, wherein theanimals have at least one of a growth deficit and a weakened immunesystem.
 13. Method for the preparation of an animal feed composition,said method comprising: admixing a composition according to claims 1with at least one or feed substance or ingredient in order to obtain ananimal feed composition according to claims
 1. 14. Method for thepreparation of an animal feed composition, said method comprising:supplementing an animal feed with free IAA or a derivative thereof inorder to obtain an animal feed composition according to claims
 1. 15.Method for raising non-human animals comprising: mixing an effectivedose of free IAA or a derivative thereof with a feed material in orderto obtain a feed composition according to claim 1, suitable for aparticular animal species; and feeding said species with the feedmaterial.
 16. A feed composition according to claim 2 comprising between100 and 1000 mg of free IAA or a derivative thereof per kilogram.
 17. Afeed composition according to claim 2 additionally comprising an enzymecapable of converting the derivative into free IAA.
 18. A methodaccording to claim 9, wherein the free IAA or a derivative thereof iscapable of increasing the serum level of insulin-like growth factor 1(IGF-1).